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LIBRARY 


THE  FATTY  ACID  RADICALS 

OF 
LIVER  LECITHIN 


Arr 


BY 

HENRY  S.  SIMMS 


TED  IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF  DOCTOR  OF  PHILOSOPHY  IN  THE  FACULTY  OF 
PURE  SCIENCE  OF:  COLUMBIA  UNIVERSITY 


NEW  YORK 
1922 


THE  FATTY  ACID  RADICALS 

OF 
LIVER  LECITHIN 


HENRY  S.  SIMMS 

\v 


Introduction 

Part  1.  The  Liver  Lecithin 

Part  2.  The  Unsaturated  Fatty  Acids  of  Liver  Lecithin 


SUBMITTED   IN  PARTIAL  FULFILLMENT  OF  THE  REQUIREMENTS   FOR  THE 

DEGREE  OF  DOCTOR  OF  PHILOSOPHY  IN  THE  FACULTY  OF 

PURE  SCIENCE  OF  COLUMBIA  UNIVERSITY 


REPRINTED  FROM 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
VOL.  XLVIII,  No.  1,  SEPTEMBER,  1921,  and  VOL.  LI,  No.  1,  MARCH,  1922 


BXCHANO* 


Introduction. 

Numerous  investigators  in  medicine  and  biology  have  come  to 
consider  lipoids  as  highly  essential  constituents  of  the  animal 
organism,  in  that  they  appear  to  be  involved  in  certain  necessary 
functions  of  the  tissues. 

The  lipoid  known  as  lecithin  occurs,  as  do  the  other  lipoids,  in 
all  animal  tissues.  The  following  structural  formula  has  been 
generally  accepted : 

CH2— O-CO-Ri 


CH— O-CO-R2 

O 

II 
CH2 — O P— O— C2H4N=  (CH3)3 

I  I 

OH  OH 

Lecithin  is  composed  of  the  five  radicals,  choline,  phosphoric 
acid,  glycerol,  and  two-  fatty  acid  radicals  (— O-CO-Ri  and 
-O-CO-R2). 

In  the  attempt  to  complete  our  knowledge  of  the  chemical 
constituents  of  lecithin,  it  seemed  advisable  to  ascertain  the 
nature  of  these  fatty  acid  radicals  with  more  care  and  accuracy 
than  had  previously  been  attempted.  As  a  result  of  the  investi- 
gations which  are  reported  in  the  following  pages,  it  has  been 
found  that  the  lecithin  from  livers  gives  on  hydrolysis  four  fatty 
acids.  Two  of  them,  stearic  and  palmitic,  are  saturated;  while 
two,  oleic  and  arachidonic,  are  unsaturated.  The  proportion  in 
which  the  unsaturated  acids  occur  has  been  found  to  vary.  Since 
the  molecules  were  shown  to  exist  in  monomolecular  form  it  is 
evident  that  lecithin  is  a  mixture  of  different  molecules  which 
vary  from  each  other  in  the  particular  fatty  acid  radicals  which 
they  contain. 


5075D6 


[Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  September,  1921,  Vol.  xlviii, 

No.  1,  pp.  185-196.] 


THE  LIVER  LECITHIN. 

BY  P.  A.  LEVENE  AND  H.  S.  SIMMS. 
(From  the  Laboratories  of  The  Rockefeller  Institute  for  Medical  Research.) 

(Received  for  publication,  July  7,  1921.) 

The  older  work  on  the  unsaturated  lipoids  of  the  liver  has  been 
reviewed  in  the  paper  of  Levene  and  Ingvaldsen.  In  the  same 
paper  a  new  analysis  of  the  liver  lecithin  was  reported.  The  new 
facts  reported  in  that  paper  related  principally  to  the  nature  of  the 
fatty  acids.  In  the  course  of  the  work  reported  by  Levene  and 
Ingvaldsen1  two  fatty  acids  were  found,  one  saturated,  and  the  other 
unsaturated,  which  analyzed  for  a  polyunsaturated  arachidic  acid. 
Since  an  acid  of  that  structure  has  never  before  been  described  in 
connection  with  lecithin,  further  corroboration  of  the  finding  seemed 
desirable.  Furthermore,  in  the  course  of  the  present  year  it  was 
shown  by  Levene  and  Rolf2  that  the  egg  lecithin  and  that  of  the  brain 
contained  two  saturated  fatty  acids.  This  result  was  obtained  by 
means  of  fractional  distillation  of  the  esters  of  the  combined  saturated 
acids.  In  light  of  this  observation  it  became  necessary  to  reinvesti- 
gate  all  lecithins  in  regard  to  the  number  of  fatty  acid  radicles, 
saturated  and  unsaturated,  present  in  their  molecule.  In  regard  to  the 
liver  lecithin  it  was  now  found  that  it  contained  two  saturated  and 
two  unsaturated  acids.3 

The  saturated  acids  are  palmitic  and  stearic.  They  were  isolated 
and  identified  by  the  same  procedure  as  described  by  Levene  and  Rolf. 

The  unsaturated  acids  are:  one,  unsaturated  stearic,  and  the 
other,  unsaturated  arachidic.  On  reduction  one  is  converted  into 
stearic  and  the  other  into  arachidic.  The  exact  degree  of  unsaturation 
of  either  one  of  the  two  acids  is  as  yet  not  known.  There  are,  however, 
indications  that  one  (arachidic)  may  be  tetra-unsaturated.  On  the 

1  Levene,  P.  A.,  and  Ingvaldsen,  T.,  /.  Biol.  Chem.,  1920,  xliii,  359. 

2  Levene,  P.  A.,  and  Rolf,  Ida  P.,  /.  Biol.  Chem.,  1921,  xlvi,  193,  353. 

3  Evidence  has  recently  been  obtained  in  this  laboratory  that  egg  lecithin 
also  contains  the  two  unsaturated  acids. 

273 


274  LIVER   LECITHIN 

addition  of  bromine  a  substance  was  obtained  which  analyzed  for  an 
octobromide  of  arachidic  acid.  However,  it  will  require  a  larger 
quantity  of  material  to  establish  the  degree  of  unsaturation  of  each 
of  the  two  acids  with  certainty.  The  presence  of  several  acids  in 
the  liver  lecithin  again  emphasizes  the  question  of  the  existence  oi 
more  than  one  lecithin. 

It  was  attempted  to  answer  this  question  by  the  molecular  weight 
estimation  of  the  hydrolecithin.  The  hydrolecithin  from  the  liver 
lecithin  has  been  prepared  essentially  according  to  Paal's  procedure. 

The  molecular  weight  of  the  substance  was  found  810  and  700 
(in  two  estimations).  The  theory  of  a  monophosphatide  requires 
809,  that  of  a  diphosphatide  1600.  Consequently,  liver  lecithin 
consists  of  a  mixture  of  monolecithins. 

In  the  course  of  the  present  work  the  process  of  preparation  of  pure 
free  lecithin  from  its  cadmium  chloride  salt  has  been  improved  so  that 
analytically  pure  substance  is  prepared  in  good  yield;  namely,  about 
50  gm.  of  free  lecithin  from  100  gm.  of  the  cadmium  chloride  salt. 

The  procedure  in  the  main  is  as  follows:  The  salt  of  lecithin  is 
dissolved  in  chloroform  and  this  solution  is  transferred  into  a  solution 
of  dry  ammonia  gas  in  dry  methyl  alcohol.  The  resulting  lecithin  is 
purified  from  the  slight  quantity  of  impurities  by  the  acetic  acid 
process  developed  by  Levene  ana  Ingvaldsen.  The  details  of  the 
procedure  are  given  in  the  experimental  part. 

EXPERIMENTAL. 

/.  Preparation  of  Pure  Lecithin. 

Various  attempts  to  produce  pure  liver  lecithin  without  converting 
it  into  the  cadmium  chloride  salt  met  with  little  success.  The 
following  method  proved  to  be  the  easiest  and  most  efficient. 

The  liver,  in  100  pound  lots,  is  minced,  dried,  and  extracted,  first 
with  acetone,  second  with  ether,  and  last  with  alcohol.  These  ex- 
tracts are  treated  separately  as  follows : 

Acetone  Extract.— This  is  allowed  to  stand  at  0°C.  over  night. 
A  precipitate  of  fat  is  deposited,  which  is  removed  by  filtration.  The 
filtrate  is  concentrated  (if  necessary)  and  the  lecithin  precipitated  by 
adding  a  saturated  solution  of  cadmium  chloride  in  alcohol  until  no 
further  precipitate  is  formed. 


P.   A.    LEVENE   AND  H.    S.    SIMMS  275 

The  residue  from  the  above  filtration  is  suspended  in  alcohol  and 
warmed  until  the  fat  is  melted.  The  mixture  is  then  cooled  over 
night.  The  fat  precipitated  on  standing  is  again  filtered  from  the 
alcoholic  solution  and  again  treated  with  alcohol  as  before.  This 
extraction  is  repeated  until  the  mother  liquor  no  longer  gives  with 
cadmium  chloride  a  precipitate  of  lecithin  cadmium  salt.  The  latter 
is  recognized  by  the  fact  that  on  dissolving  in  a  small  amount  of 
moist  ether  it  is  again  precipitated  by  the  addition  of  an  excess  of 
acetone.  From  three  to  seven  extractions  may  be  required.  The 
alcoholic  mother  liquors  are  then  precipitated  with  cadmium  chloride. 

Ether  Extract. — This  is  concentrated  to  a  small  volume  and  allowed 
to  stand  at  0°C.,  when  a  precipitate  consisting  of  fat  and  cerebrosides 
is  formed.  The  precipitate  is  extracted  with  ether.  The  ethereal 
extract  is  added  to  the  original  filtrate  and  cooled  once  more  to  per- 
mit the  separation  of  the  cerebrosides  which  the  solution  may  still 
contain.  After  filtering,  the  combined  mother  liquors  are  concen- 
trated and  treated  with  alcohol  to  separate  the  lecithin  from  cephalin. 
The  alcoholic  liquors  are  then  treated  with  cadmium  chloride. 

Alcoholic  Extract. — This  is  likewise  concentrated  and  cooled  to 
remove  cerebrosides,  the  mother  liquor  being  decanted  if  possible, 
otherwise  filtered,  or  centrifuged  if  necessary. 

The  cerebrosides  are  again  extracted  with  warm  ether.  The 
extract  is  cooled  and  centrifuged.  The  alcoholic  and  ethereal  liquors 
are  then  treated  with  cadmium  chloride. 

Treatment  of  Cadmium  Chloride  Salts. 

It  is  necessary  to  allow  the  cadmium  chloride  precipitate  of  lecithin 
to  stand  at  least  half  an  hour  until  it  is  sufficiently  coagulated  to 
permit  filtration.  The  filtered  material,  which  is  not  quite  dry,  is 
transferred  to  a  large  beaker  or  precipitating  jar  and  stirred  up  with 
a  large  volume  of  cold  acetone.  If  the  acetone  liquor  turns  dark 
from  dissolved  material  the  suspension  is  allowed  to  settle,  the  liquor 
decanted  off,  and  more  cold  acetone  is  added.  Finally  the  material 
is  filtered  by  suction. 

This  material  is  purified  in  two  steps:  the  one  is  the  "ether  crys- 
tallization," the  other  is  the  " toluene-ether"  process.1  It  is  a  matter 


276  LIVER  LECITHIN 

of  judgment  as  to  which  shall  be  used  first  and  the  number  of  times 
which  each  should  be  repeated.  The  aim  is  to  obtain  a  white  granular 
material  which  filters  quickly. 

The  ether  crystallization  consists  in  dissolving  the  cadmium  chloride 
salt  in  warm  ether,  water  being  added,  a  few  drops  at  a  time,  until  the 
suspended  material  goes  into  solution.  An  excess  of  water  hinders 
the  solution  of  the  larger  particles.  The  solution  is  allowed  to  stand 
over  night,  or  longer,  at  0°C.  The  substance  should  separate  in  a 
granular  form,  easily  filterable  by  suction.  If  it  forms  a  pasty  solution 
not  easily  filtered,  time  and  material  will  be  wasted  in  attempting 
a  filtration.  Another  precipitation  with  acetone  should  remove 
impurities  which  interfere  with  the  process. 

This  purification  removes  not  only  the  fats  and  oils  but  also  takes 
out  most  of  the  cephalin  present.  Since  the  cadmium  chloride  salt 
is  itself  slightly  soluble  in  cold  ether,  some  of  the  material  may  be 
lost  in  purification.  Hence  the  following  precautions  are  necessary. 
1.  Excess  of  ether  is  to  be  avoided.  With  very  impure  material  it  is 
more  advisable  to  repeat  the  purification  several  times  with  small 
quantities  of  solvent  than  to  use  a  large  excess  at  one  time.  The 
amount  of  ether  filtered  off  should  not  be  more  than  twice  the  volume 
of  the  residue.  2.  The  filtered  material  should  not  be  washed  with 
ether,  but  should  be  filtered  as  quickly  as  possible  until  the  solvent 
runs  very  slowly.  3.  In  case  the  material  fails  to  filter  properly,  it 
should  be  transferred  to  a  beaker,  warmed  slightly  until  dissolved, 
precipitated  with  acetone,  purified  by  the  toluene  ether  method, 
and  subsequently  passed  through  the  ether  crystallization  process. 
4.  The  filtration  should  be  carried  out  in  the  cold. 

The  toluene-ether  process  consists  in  dissolving  the  cadmium  chloride 
salt  in  a  minimum  volume  of  toluene  (adding  a  slight  amount  of 
water  if  necessary).  If  the  toluene  fails  to  dissolve  all  the  material 
the  residue  should  be  centrifuged  off.  The  solution  is  then  treated 
with  4  volumes  of  ether  containing  1  per  cent  water.  The  solution  is 
cooled  to  0°C.  over  night  and  filtered. 

The  latter  method  gives  larger  yields  but  removes  less  of  the 
cephalin  and  other  impurities.  It  probably  removes  impurities  not 
taken  out  by  the  former  method,  hence  the  cadmium  chloride  com- 
pound should  be  purified  by  both  methods. 


P.   A.    LEVENE   AND   H.    S.    SIMMS  277 

Experience  shows  that  in  the  case  of  liver  lecithin  the  toluene- 
ether  method  should  precede  the  ether  crystallization  method  of 
purification  of  the  cadmium  chloride  salt  in  order  to  obviate  difficulty 
in  filtering  from  the  ether. 

One  purification  by  each  method  should  be  sufficient  to  give  almost 
white  dry  material  with  an  amino  content  of  less  than  3  per  cent  of 
the  total  nitrogen  present.  Such  a  product  may  be  converted  into 
free  lecithin. 

Conversion  of  the  Cadmium  Chloride  Compound  into  Free  Lecithin. 

The  cadmium  chloride  salt  is  dissolved  in  chloroform  and  is  con-  ' 
verted  into  free  lecithin  by  means  of  a  solution  of  ammonia  in  methyl 
alcohol.  100  gm.  of  the  cadmium  chloride  salt  are  dissolved  in  300 cc. 
of  warm  chloroform  and  poured  into  400  cc.  of  methyl  alcohol  con- 
taining 20  gm.  of  ammonia  gas.  This  is  added  slowly  with  rapid 
stirring.  The  product  of  reaction  is  allowed  to  stand  a  short  time 
before  filtering.  The  precipitate  may  be  filtered  off  through  a  folded 
filter  paper.  The  chloroform  methyl  alcohol  solution  of  lecithin  is 
then  concentrated  under  diminished  pressure.  Near  the  end  of  the 
concentration  the  material  foams  considerably  for  a  short  time  and 
then  the  foaming  subsides.  The  vacuum  concentration  should  be 
carried  out  at  a  low  temperature.  If  during  the  operation  a  precipi- 
tate of  fat  settles  out  this  should  be  filtered  off.  The  remaining  leci- 
thin is  practically  free  from  solvent.  It  is  dissolved  in  a  minimum 
(5  to  10  cc.)  of  glacial  acetic  acid.  This  is  poured  into  800  cc.  of 
boiling  hot  acetone,  stirred,  and  allowed  to  cool  to  room  temperature. 
A  very  small  dark  precipitate  (1  to  2  gm.)  settles  out.  The  superna- 
tant liquid  is  decanted  or  filtered.  The  precipitate  is  slightly  soluble 
in  ether  and  insoluble  in  acetone  but  somewhat  soluble  in  ethyl 
alcohol  and  more  soluble  in  methyl  alcohol. 

No.  126. 

0.0154  gm.  of  substance  gave  on  combustion  0.0954  gm.  6f  H;0,  0.2315 
gm.  of  C02,  and  0.0114  gm.  of  ash. 

0.1910  gm.  of  substance  used  for  Kjeldahl  nitrogen  determination  required 
3.90  cc.  of  0.1  N  acid  corresponding  to  0.00546  gm.  of  N. 

0.2865  gm.  of  substance  gave  0.0390  gm.  of  Mg2P207. 


278  LIVER    LECITHIN 

C44Hg7O9NP.     Calculated.     C  65.59,  H  10.89,  N  1.74,  P  3.86. 
Found.  C  61.16,  H  10.34.  N  2.92,  P  3.88. 

It  'contains  10  per  cent  amino  nitrogen. 

No.  124. 

1.5  gm.  of  substance  were  hydrolyzed  with  HC1,  neutralized,  concentrated, 
and  made  up  to  15  cc. 

5  cc.  of  this  solution  required  for  Kjeldahl  nitrogen  determination  5.60  cc.  of 
0.1  N  HC1. 

2  cc.  of  this  solution  for  Van  Slyke  determination  gave  0.57  cc.  of  N2  at  T  = 
27°,  P  =  762.2  mm. 

Amino  N        10 
Total   N  "  100 

The  liquors  are  then  cooled  in  a  freezing  mixture  to  —  5°C.  Fre- 
quently at  this  phase  a  second  small  precipitate  settles  out.  A  sample 
of  this  material  analyzed  as  follows: 

No.  122. 

0.1024  gm.  of  substance  gave  on  combustion  0.1098  gm.  of  H^O,  0.2216  gm. 
of  CO 2,  and  0.0118  gm.  of  ash. 

0.1832  gm.  of  substance  for  Kjeldahl  nitrogen  determination  required  3.10  cc. 
of  0.1  N  acid  corresponding  to  0.00434  gm.  of  N. 

0.2748  gm.  of  substance  gave  0.0464  gm.  of  Mg2P2O7. 

C44H87O9NP.     Calculated.     C  65.59,  H  10.89,  N  1.74,  P  3.86. 
Found.  C  59.50,  H  12.10,  N  2.37,  P  4.75. 

The  mother  liquors  are  concentrated  under  diminished  pressure 
until  all  the  ether  and  most  of  the  acetic  acid  are  removed.  Water  is 
added  a  little  at  a  time  and  the  material  is  shaken  or  stirred  until  a 
thick  emulsion  of  a  light  brown  color  is  formed.  This  is  poured  into 
800  to  1,000  cc.  of  acetone,  chilled  down  to  —  5°C.  It  is  carefully 
stirred  and  allowed  to  stand  at  0  to  —  5°C.  over  night,  when  it  is 
transferred  to  a  crystallizing  dish  and  washed  free  from  excess  water 
by  stirring  with  cold  dry  acetone.  The  acetone  is  decanted  off  and  the 
lecithin  dried  in  a  vacuum  desiccator. 

From  40  to'  45  gm.  of  pure  material  may  be  obtained  from  100  gm. 
of  cadmium  chloride  salt.  (Theoretical  yield,  81  to  82  gm.)  Several 
samples  have  been  analyzed.  They  differed  little  one  from  another  in 
their  elementary  composition.  The  analysis  of  one  of  thejn  is  as 
follows: 


P.   A.    LEVENE    AND   H.    S.    SIMMS  279 

No.  119. 

0.0996  gm.  of  substance  gave  on  combustion  0.0994  gm.  of  H20,  0.2768  gm. 
of  CO  2,  and  0.0090  gm.  of  ash. 

0.1798  gm.  of  substance  required  2.40  cc.  of  0.1  N  acid,  corresponding  to  0.00336 
gm.  of  N. 

0.2697  gm.  of  substance  gave  0.0390  gm.  of  Mg2P207. 

C44H87O9NP.*  Calculated.     C  65.59,  H  10.89,  N  1.74,  P  3.86. 
Found.  C  64.83,  H  11.16,  N  1.87,  P  4.03. 

*  This  formula  represents  material  consisting  of  equal  parts  of  two  lecithins, 
each  one  containing  two  of  the  four  fatty  acids. 

II.  The  Fatty  Acids  of  Lecithin. 

For  the  preparation  of  fatty  acids  from  lecithin,  the  material  was 
hydrolyzed  8  to  15  hours  with  10  parts  of  10  per  cent  HC1.  The 
fatty  acids  on  cooling  appeared  as  a  semisolid  cake.  They  were 
dissolved  in  methyl  alcohol  and  precipitated  in  the  presence  of  am- 
monium hydroxide  with  a  nearly  equal  weight  of  lead  acetate  dissolved 
in  a  minimum  quantity  of  water.  After  freezing,  the  mother  liquors 
were  filtered  off.  The  lead  salts  which  contained  both  the  saturated 
and  unsaturated  fatty  acids  were  extracted  repeatedly  with  boiling 
ether  until  further  extraction  produced  only  slight  precipitate  with 
hydrochloric  acid. 

The  ether  solution  then  contained  the  lead  salts  of  the  unsaturated 
acids  while  the  ether-insoluble  material  consisted  of  the  lead  salts  of 
the  saturated  acids.  Both  fractions  were  decomposed  with  HC1, 
dissolved  in  ether,  washed  with  water,  dried,  and  the  solvent  evapo- 
rated off. 

A  lot  of  528  gm.  of  the  lecithin  cadmium  chloride  free  from  amino 
nitrogen  was  hydrolyzed  with  10  per  cent  solution  of  hydrochloric 
acid.  The  yield  of  fatty  acids  was  223  gm. 

Unsaturated  Fatty  Acids. 

These  were  obtained  by  extracting  the  lead  salt  first  by  means  of 
acetone  and  then  by  means  of  ether.  Each  extract  was  worked  over 
separately.  The  acetone  extract  was  concentrated  and  the  residue 
thus  obtained  extracted  with  ether.  From  both  of  these  fractions  the 
acids  were  liberated  and  reconverted  into  the  lead  salts.  These  were 


280  LIVER  LECITHIN 

again  purified  and  again  converted  into  the  free  acids.     A  sample  of 
the  acids  gave  the  following  iodine  and  hydrogen  values: 

0.2907  gm.  of  substance  absorbed  0.435  gm.  of  iodine  by  the  Wijs  method. 

0.5141  gm.  of  substance  reduced  by  Paal's  method  absorbed  67  cc.  of  H2 
in  3  hours  at  17°C.,  759  mm.  pressure,  or  1.103  gm.  of  H2  per  100  gm.  of 
substance. 

Ci8H34O2.    Calculated.    Iodine  value    91,  Hydrogen  number  0.721. 

Ci8H3202.     Calculated.          "        "     182,          "  "       1.447. 

Found.  "        "     154,  "       1.103. 

It  was  later  found  that  this  material  consisted  of  two  fatty  acids, 
hence  it  is  possible  that  one  was  a  singly  unsaturated,  and  that  the 
other  contained  two  or  more  double  bonds. 

The  free  fatty  acids  were  finally  reduced  by  Paal's  method.  The 
samples  of  reduced  acids  obtained  from  each  fraction  analyzed  as 
follows : 

No.  84  (material  obtained  from  the  acetone  extract  of  the  lead  salts). 

0.1020  gm.  of  substance  gave  on  combustion  0.1198  gm.  of  H^O  and  0.2830 
gm.  of  CC>2. 

No.  85  (material  obtained  from  the  ether  extract  of  the  lead  salts) 

0.1012  gm.  of  substance  gave  on  combustion  0.1190  gm.  of  H2O  and  0.2828 
gm.  of  CO 2. 

No.  84.    C  75.96,  H  13.19. 
"  85.    C  76.21,  H  13.15. 

Since  the  two  fractions  proved  practically  of  identical  elementary 
composition  they  were  combined  and  converted  into  the  methyl  esters. 
These  were  freed  from  adhering  sulfuric  acid  by  washing  with  water 
and  finally  by  recrystallization  from  methyl  alcohol.  They  were  then 
fractionated  by  distilling  at  a  pressure  of  1  to  2  mm. 

The  following  fractions  were  obtained. 

A 182-185°C. 

B '. 175-185°C. 

C 182-195°C. 

D 185-203°C. 

Fractions  A  and  D  were  redistilled  and  the  following  fractions  were 
obtained. 


P.    A.    LEVENE    AND    H.    S.    SIMMS  281 

From  A        Ai 158-165°C. 

A2 170-182°C. 

From  D        Dx 182-192°C. 

D2 187-197°C. 

For  identification,  the  esters  were  saponified  with  an  alcoholic  solu- 
tion of  sodium  hydroxide.  The  acids  were  liberated  and  converted 
into  the  lead  salts.  The  acids  were  again  liberated  from  the  lead  salts 
and  analyzed.  Fraction  Aa  corresponded  apparently  to  pure  stearic 
acid. 

Analysis  101. 

0.1024  gm.  of  substance  gave  on  combustion  0.1186  gm.  of  H^O  and  0.2860 
gm.  of  CO  2. 

0.8950  gm.  of  substance  in  a  molecular  weight  determination  required  6.50  cc. 
of  0.5  N  NaOH. 

Ci8H36O2.     Calculated.     C  75.93,  H  12.76. 
Found.  C  76.16,  H  12.96. 

Molecular  weight  was  275,  that  of  stearic  acid  is  284. 

The  substance  melted  at  70.5-71°.C,  stearic  acid  melts  at  70-7l°C. 

When  this  was  mixed  with  some  very  pure  stearic  acid  melting  at 
74°C.,  the  mixture  melted. at  74°C. 
Fraction  D2  apparently  corresponded  to  pure  arachidic  acid. 

Analysis  100. 

0.1000  gm.  of  substance  gave  on  combustion  0.1166  gm.  of  H20  and  0.2822 
gm.  of  CO  2. 

0.9760  gm.  of  substance  neutralized  6.75  cc.  of  9.5  N  NaOH. 

C20H4oO2.    Calculated.    C  76.95,  H  12.91. 
Found.  C  76.97,  H  13.24. 

Molecular  weight  was  314,  that  of  arachidic  acid  is  313. 

The  substance  melted  at  75.5-76°C.,  arachidic  acid  melts  at  75-77°C. 

When  this  was  mixed  with  some  pure  arachidic  acid  melting  at 
75°C.,  the  mixture  melted  at  75°C. 

Saturated  Fatty  Acids. 

The  lead  salts  which  were  insoluble  in  acetone  and  ether  were  con- 
verted into  free  acids.  These  were  twice  esterified  with  methyl 
alcohol.  The  mixture  of  methyl  esters  thus  obtained  was  distilled  at 
a  pressure  of  1  to  2  mm.  into  the  following  fractions: 


282  LIVER   LECITHIN 

a 160-163°C. 

b ; 159-167°C. 

c 158-1 72°C. 

d 170-180°C. 

and  residue. 

Fractions  a  and  d  were  redistilled  as  follows. 

From  a          at 156-162°C. 

a2 Residue. 

From  d         di 180-183°C. 

d2 182-188°C. 

Fraction  ai  apparently  corresponded  to  pure  palmitic  acid. 

Analysis  92. 

0.1009  gm.  of  substance  gave  on  combustion  0.1220  gm.  of  H20  and  0.2802 
gm.  of  CO 2. 

0.8168  gm.  of  substance  neutralized  6.14  gm.  of  0.5  N  NaOH. 

CieHssO*.     Calculated.     C  74.92,  H  12,58. 
Found.  C  75.09,  H  12.98. 

Molecular  weight  was  found  to  be  266,  palmitic  acid  had  a  molecular  weight 
of  256. 

The  melting  point  was  62°C.,  palmitic  acid  melts  at  63-64°C. 

When  this  was  mixed  with  some  pure  palmitic  acid  melting  at 
64°C.,  the  mixture  melted  at  63°C. 

Fraction  d2  apparently  corresponded  to  pure  stearic  acid. 

Analysis  94. 

0.1009  gm.  of  substance  gave  on  combustion  0.1220  gm.  of  H20  and  0.2802 
gm.  of  CO  2. 

0.6686  gm.  of  substance  neutralized  4.82  cc.  of  0.5  N  NaOH. 

C18H86O2.    Calculated.     C  75.93,  H  12.76. 
Found.  C  75.72,  H  13.53. 

Molecular  weight  was  278,  stearic  acid  had  a  molecular  weight  of  284. 
The  substance  melted  at  7l°C.,  stearic  acid  melts  at  70-7l°C. 

When  this  was  mixed  with  a  sample  of  very  pure  stearic  acid  melting 
at  74°C.  the  mixture  melted  at  74°C. 

///.  Bromine  Addition  Products  of  the  U maturated  Acids. 

An  attempt  was  made  to  separate  and  to  characterize  th:e  individ- 
ual unsaturated  acids  by  preparing  the  bromine  addition  products. 


P.    A.    LEVENE    AND    H.    S.    SIMMS  283 

40  gm.  of  pure  lecithin,  which  had  been  prepared  from  the  cadmium 
chloride  salt  as  described  above,  were  used. 

No.  119. 

0.0996  gm.  of  substance  gave  on  combustion  0.0994  gm.  of  H20,  0.2768  gm. 
of  CO 2,  and  0.0090  gm.  of  ash. 

0.1798  gm.  of  substance  required  2.40  cc.  of  0.1  N  acid,  corresponding  to  0.00336 
gm.  of  N. 

0.2697  gm.  of  substance  gave  0.0390  gm.  of  Mg2P2O7. 

C44H87O9NP.    Calculated.    C  65.59,  H  10.89,  N  1.74,  P  3.86. 
Found.  C  64.83,  H  11.16,  N  1.87,  P  4.03. 

This  was  hydrolyzed  with  a  10  per  cent  solution  of  hydrochloric 
acid,  the  acids  were  dissolved  in  ether,  washed  with  water,  dried, 
and  the  ether  evaporated  off.  The  iodine  number  of  the  mixed 
acids  was  91. 

0.2457  gm.  of  substance  absorbed  0.232  gm.  of  iodine  by  the  Wijs  method. 

Average  molecular  weight  of  280.    Calculated.    Iodine  value  91. 

Found.  "          "    91. 

The  acids  were  converted  into  the  lead  salts,  the  unsaturated  acids 
extracted  with  ether  and  converted  into  the  free  acids.  These  were 
dissolved  in  18-30°  petrolic  ether  and  brominated  at  0°C.  with  3  cc. 
of  bromine  dissolved  in  petrolic  ether. 

On  freezing  to  -10°  a  precipitate  was  obtained.  The  mother  liquor 
was  concentrated  and  again  cooled  to  — 10°.  The  combined  precipi- 
tate was  recrystallized  from  petrolic  ether  and  then  recrystallized 
from  ethyl  ether. 

This  gives  three  fractions:  A,  the  petrolic  ether-soluble  fraction: 
B,  the  fraction  insoluble  in  petrolic  ether  but  soluble  in  ethyl  ether; 
and  C,  the  fraction  insoluble  in  both  solvents.  This  last  fraction 
contains  the  material  having  most  bromine  (namely,  the  hexabromides 
and  octobromides,  if  present).  The  first  fraction  should  be  largely 
dibromides  while  the  tetrabromides  should  predominate  in  the  fraction 
insoluble  in  petrolic  ether  but  soluble  in  ethyl  ether. 

Fraction  C  (the  material  insoluble  in  both  solvents)  was  recrystal- 
lized from  ethyl  ether;  the  yield  was  1  gm.  In  an  open  tube  melting 
point  determination  it  darkened,  turning  black  at  200°C.  It  con- 
tracted at  240°C.  and  decomposed  at  243°C.  In  a  closed  tube  it  con- 
tracted at  239°C.  and  melted  without  decomposition  at  243°C.  This 
analyzed  as  follows: 


284  LIVER    LECITHIN 

No.  129. 

0.2012  gm.  of  substance  gave  0.2936  gm.  of  AgBr. 

This  would  indicate  a  hexabromide. 

C2oH34O2Br6.    Calculated  for  hexabromarachidic  acid.       i.O. 
Ci8H3oO2Br8.    Calculated  for  hexabromstearic  acid.        63.2. 
Found.  62.11. 

The  material  was  recrystallized  from  ether.  On  heating  in  an 
open  tube  it  darkened  at  180-200°C.,  contracted  at  240°C.,  and 
decomposed  at  244°C. 

It  analyzed  as  follows : 

Analysis  130. 

0.1068  gm.  of  substance  gave  0.1670  gm.  of  AgBr. 

This  corresponds  more  closely  to  an  octobromide. 

CaoH^OaBrg.    Calculated  for  octobromarachidic  acid.    67.80. 
Found.  66.55. 

There  was  not  sufficient  material  for  further  treatment. 
IV.  Hydrolecithin  from  Liver  Lecithin. 

For  the  preparation  of  hydrolecithin  10  gm.  of  pure  liver  lecithin 
free  from  amino  nitrogen  were  used  (Analysis  119  given  above). 
This  was  reduced  by  Paal's  method.  The  hydrolecithin  produced  was 
recrystallized  twice  from  acetone  and  once  from  methyl  ethyl  ketone. 

This  analyzed  as  follows: 

No.  128. 

0.1074  gm.  of  substance  gave  on  combustion  0.1082  gm.  of  HgO,  0.2554  gm. 
of  C02,  and  0.0098  gm.  of  ash. 

0.1926  gm.  of  substance  for  Kjeldahl  nitrogen  determination  required  2.40  cc. 
of  0.1  N  acid,  corresponding  to  0.00336  gm.  of  N. 

0.2839  gm.  of  substance  gave  0.0400  gm.  of  Mg2P207. 

C44H3iO9NP.    Calculated.     C  65.30,.H  11.33,  N  1.73,  P  3.84. 
Found.         '  C  65.03,  H  11.29,  N  1.74,  P  3.86. 

A  molecular  weight  determination  was  made  as  follows: 

1.036  gm.  of  substance  raised  the  boiling  point  of  16  gm.  methyl  alcohol  0.07l°C. 
0.964  gm.  of  substance  raised  the  boiling  point  of  16  gm.  methyl  alcohol  0.077°C. 

C44H9iO»NP.     Calculated.    Molecular  weight  809. 

Found.    First  .determination     810 

Second       "  700. 


Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
Vol.  LI,  No.  1,  March,  1922 


THE  UNSATURATED  FATTY  ACIDS  OF  LIVER  LECITHIN. 

BY  P.  A.  LEVENE  AND  H.  S.  SIMMS. 

(From  the  Laboratories  of  The  Rockefeller  Institute  for  Medical  Research.} 

(Received  for  publication,  January  3,  1922.) 

In  previous  communications  on  liver  lecithin  results  were 
reported  showing  that  the  unsaturated  fatty  acids  obtained  on 
hydrogenation  gave  stearic  and  arachidic  acids.  It  was  further 
reported  that  the  iodine  number  of  the  mixed  unsaturated  fatty 
acids  was  154,  and  that  the  mixed  unsaturated  acids  on  bromina- 
tion  formed  a  bromide,  which  on  the  basis  of  its  bromine  content 
and  of  its  melting  point  seemed  to  be  the  octobromarachidic 
acid. 

From  these  findings  it  was  concluded  that  liver  lecithin  con- 
tained more  than  one  unsaturated  fatty  acid,  and  that  the  indi- 
vidual acids  differed  in  the  degree  of  their  unsaturation.  It  then 
seemed  suggestive  that  the  acid  of  higher  unsaturation  was  the 
tetra  unsaturated  arachidic  acid.  Since  there  was  no  accurate 
knowledge  either  as  to  the  number  of  the  unsaturated  acids  present 
in  the  mixture  or  as  to  the  degree  of  their  unsaturation,  there  was 
no  indication  as  to  the  ratio  of  the  individual  acids.  In  order  to 
complete  our  knowledge  of  the  unsaturated  fatty  acids,  it  became 
necessary  to  find:  (1)  the  number  of  the  unsaturated  fatty  acids 
present  in  the  liver  lecithin;  (2)  the  exact  nature  of  the  acids  of 
higher  unsaturation;  and  (3)  the  ratio  of  the  individual  fatty 
acids. 

In  the  course  of  the  present  work  it  was  found  that  liver  lecithin 
yields  only  two  unsaturated  fatty  acids,  oleic  and  arachidonic1 
acids,  and  of  these  oleic  acid  predominates. 

1  Since  the  tetra  unsaturated  arachidic  acid  is  called  arachidonic  acid  by 
Lewkowitsch  (Lewkowitsch,  J.,  Chemical  technology  and  analysis  of  oils, 
fats  and  waxes,  London,  4th  edition,  1909,  i,  211)  this  name  will  be  adopted 
by  the  present  writers. 

285 


286  Fatty  Acids  of  Liver  Lecithin 

In  connection  with  the  finding  of  arachidonic  acid  as  a  con- 
stituent of  liver  lecithin,  it  is  interesting  to  recall  the  work  of 
Hartley,2  done  under  the  direction  of  Leathes.  On  oxidation  of 
the  unsaturated  acids  from  liver  fat,  including  phosphatides,  this 
author  isolated  an  unsaturated  fatty  acid  which  was  oxidized 
to  a  tetrahydroxyarachidic  acid.  Furthermore,  on  bromination 
of  the  same  fatty  acids  Hartley  obtained  a  bromide,  having  a 
bromine  content  only  0.7  per  cent  higher  than  that  required  by 
theory  for  octobromarachidic  acid. 

The  present  writers,8  and  Levene  and  Ingvaldsen,4  have  pre- 
viously reported  that  on  hydrogenation  of  the  unsaturated  acids 
of  liver  lecithin,  arachidic  acid  was  isolated.  They  have  also 
obtained  from  liver  lecithin  a  bromide,  which  on  analysis  gave  a 
bromine  value  about  1  per  cent  lower  than  that  required  by  theory 
for  the  octobromarachidic  acid.  In  the  course  of  the  present  work 
an  octobromarachidic  acid  was  isolated  which  gave  correct  analyt- 
ical values.  This  acid  was  then  reconverted  into  a  tetra  unsatu- 
rated arachidic  acid,  which  on  hydrogenation  was  converted  into 
arachidic  acid.  From  the  product  of  bromination  of  the  unsatu- 
rated fatty  acids,  fractions  were  obtained  which  analytically 
resembled  the  hexa-  and  tetrabromides.  On  purification  of  these 
fractions  only  the  octobromide  was  isolated  in  pure  form.  Hence 
present  day  evidence  does  not  permit  us  to  assume  the  presence  of 
more  than  one  poly  unsaturated  fatty  acid  in  lecithin.  On  the  other 
hand,  from  the  more  soluble  fraction  of  the  bromination  product 
a  substance  was  obtained  which  yielded  unsaturated  fatty  acids 
with  an  iodine  number  of  107.  This  is  not  far  removed  from 
that  required  by  oleic  acid  which  has  an  iodine  number  of  90. 
From  this  fraction  on  hydrogenation  pure  stearic  acid  was  isolated. 

Assuming  that  only  two  unsaturated  acids,  arachidonic  and 
oleic  acids,  are  present  in  the  lecithin  fraction,  and  taking  into 
consideration  the  fact  that  the  iodine  number  of  the  mixed  un- 
saturated acids  of  this  lecithin  is  196,  it  becomes  easy  to  calculate 
the  ratio  of  the  two  acids  from  the  equation  90z  +  335?/  =  196 
(x  -f-  y).  It  follows  that  the  ratio  is  approximately  1.3  parts  of 
oleic  acid  to  1  part  of  arachidonic  acid. 

1  Hartley,  P.,  J.  Physiol.,  1908-09,  xxxviii,  353. 

1  Levene,  P.  A.,  and  Simms,  H.  S.,  J.  Biol.  Chem.,  1921,  xlviii,  185. 

4  Levene,  P.  A.,  and  Ingvaldsen,  T.,  J.  Biol.  Chem.,  1920,  xlifr,  359. 


P.  A.  Levene  and  H.  S.  Simms  287 

The  lecithin  referred  to  above  was  obtained  from  the  acetone 
extract  of  the  liver.  From  the  ethereal  extract  there  was  ob- 
tained a  lecithin  of  lower  unsaturation.  The  iodine  number  of 
the  unsaturated  acids  was  136.  This  corresponds  to  4.3  parts  of 
oleic  acid  to  1  part  of  arachidonic  acid. 

Bearing  in  mind  the  molecular  weight  estimation  of  dihydro- 
lecithin  reported  in  the  previous  communication,  namely  810, 
one  is  justified  in  concluding  that  the  liver  contains  several  leci- 
thins and  that  the  oleyl  lecithins  predominate. 

EXPERIMENTAL. 

Preparation  of  Material. 

Beef  livers  in  100  Ib.  lots  were  minced,  dried,  and  extracted, 
first  with  acetone,  then  with  ether,  and  last  with  alcohol.  These 
extracts  were  treated  by  the  methods  described  in  an  earlier  paper. 
A  cadmium  chloride  salt  of  pure  lecithin  was  obtained  which  was 
free  from  amino  nitrogen. 

Analysis  5.  1.0  gin.  substance  was  hydrolyzed  with  HC1,  neutralized, 
concentrated,  and  made  up  to  15  cc. 

2  cc.  of  this  solution:  no  N2  (Van  Slyke). 

5    "    "    "          "       :  1.95  cc.  of  0.1  N  HOI  (Kjeldahl). 

Amino  N 

X  100  =  0  per  cent  ammo  nitrogen. 

Total  N 

The  Unsaturated  Fatty  Adds. 

765  gm.  of  the  above  mentioned  material  were  hydrolyzed  with 
10  per  cent  HC1  for  15  hours.  The  fatty  acids  which  separated 
were  dissolved  in  ether  and  washed  with  water  until  free  from 
mineral  acid.  The  ether  solution  was  dried  and  concentrated  to 
about  1.5  liters.  This  solution  was  allowed  to  stand  at  0°C.  over 
night.  A  white  precipitate  settled  out.  This  was  filtered  off 
and  recrystallized  twice  from  ether  and  once  from  dry  acetone. 
It  analyzed  as  follows: 

Analysis  15.    0.1019  gm.  substance:  0.1184  gm.  H2O  and  0.2834  gm.  CO2. 
C18Hj802.    Calculated.    C  75.93,  H  12.76. 
Found.  "75.84,   "  13.00. 


288  Fatty  Acids  of  Liver  Lecithin 

The  ether  mother  liquors  from  which  this  stearic  acid  had  been 
separated  were  then  concentrated  to  dryness  under  diminished 
pressure.  The  residue  was  dissolved  in  methyl  alcohol,  treated 
with  lead  acetate  solution,  and  made  alkaline  with  ammonium 
hydroxide.  On  cooling  this  solution  to  —  5°C.  the  lead  salts  of  the 
fatty  acids  settled  out.  These  were  filtered  off  and  extracted 
repeatedly  with  ether  until  ether  would  no  longer  extract  material 
giving  a  precipitate  with  hydrochloric  acid. 

The  combined  ether  extracts  of  the  lead  salts  were  treated  with 
HC1  to  convert  the  salts  into  free  fatty  acids.  The  ether  solution 
was  then  washed  with  water  until  free  from  mineral  acid  and 
dried  with  sodium  sulfate.  The  ether  was  removed  by  distilla- 
tion under  diminished  pressure.  The  residue  of  unsaturated 
fatty  acids  weighed  160  gm.  The  iodine  number  was  obtained. 

0.2281  gm.  of  substance  absorbed  0.453  gm.  of  iodine  by  the  Wijs  method. 

Average  molecular  weight  290. 

Calculated  (two  double  bonds) .     Iodine  number  175. 

Found.  "  "       196. 

These  unsaturated  acids  were  dissolved  in  glacial  acetic  acid  and 
brominated  with  a  25  per  cent  solution  of  bromine  in  glacial 
acetic  acid.  The  temperature  was  kept  as  low  as  possible  with- 
out freezing  the  acetic  acid. 

The  bromine  solution  was  added  from  a  special  vacuum  jacket 
burette  constructed  for  the  purpose.  This  consisted  of  two 
tubes  of  Pyrex  glass  sealed  together  at  both  ends,  with  a  stop- 
cock attached.  The  lower  end  of  the  inner  tube  consisted  of 
spiral  capillary  tubing  to  take  up  the  thermal  expansion.  The 
space  between  the  outer  and  inner  tubes  was  evacuated  and  sealed. 
Bromine  solution  in  this  burette  could  be  kept  cool  longer  than  in 
an  ordinary  burette. 

The  acids  were  brominated  in  5  to  10  gm.  lots  and  allowed  to 
stand  over  night  with  a  slight  excess  of  bromine.  A  yellow  pre- 
cipitate formed  which  was  filtered  off. 

The  precipitate  (Fraction  A)  was  expected  to  contain  the  higher 
bromides  and  the  acetic  acid  mother  liquor  (Fraction  B),  the 
lower  bromides. 


P.  A.  Levene  and  H.  S.  Simms  289 

Fraction  A,  the  Fraction  Insoluble  in  Glacial  Acetic  Acid. 

Identification  of  Arachidonic  Acid. — The  precipitate  was  ex- 
tracted repeatedly  with  ether  until  it  no  longer  contained  any 
ether-soluble  material.  This  residue  analyzed  as  follows: 

Analysis  17.    0.1066  gm.  substance:  0.0356  gm.  H2O  and  0.0998gm.COz. 

0.1996     "  "         :0.3166gm.  AgBr. 

C2oH32O2Br8.    Calculated.    C  25  43,  H  3.42,  Br  67.72. 

Found.  "  25.53,  "  3.73,  "    67.57. 

In  melting  point  determination  this  material  darkened,  melted, 
and  decomposed  at  245°C.  (A  slightly  less  pure  sample  of  this 
material  previously  obtained  contracted  at  240°C.  and  decom- 
posed at  244°C.  while  it  began  to  darken  below  200°C.) 

20  gm.  of  the  above  material  were  obtained.  10  gm.  of  this 
were  suspended  in  dry  methyl  alcohol  and  reduced  with  zinc 
dust  and  dry  hydrochloric  acid  gas.  The  methyl  alcohol  was 
filtered,  diluted  with  water,  and  shaken  repeatedly  with  gasoline. 
The  gasoline  solution  was  then  shaken  with  water,  dried,  and 
evaporated  to  dryness  under  diminished  pressure.  The  iodine 
number  of  the  resulting  material  was  obtained. 

0.0492  gm.  substance  absorbed  0.150  gm.  iodine  by  the  Wijs  method. 
C2oH32O2.    Calculated.    Iodine  number  335. 
Found.  "  "      305. 

This  unsaturated  material  was  reduced  with  hydrogen  in  al- 
coholic solution  by  the  method  of  Paal.  The  solution,  after  being 
filtered,  was  concentrated  and  cooled  to  —  5°C.  The  reduced 
acid  separated  out.  This  was  recrystallized  three  times  from  dry 
acetone.  It  analyzed  as  follows: 

Analysis  34.    0. 1034  gm.  substance:  0. 1176  gm.H2O  and  0. 2912  gm.  CO-,. 
CzoEUOa.    Calculated.    C  76.85,  H  12.91. 
Found.  "  76.81,  "  12.72. 

Identification  of  Oleic  Acid. — It  was  stated  above  that  the 
octobromarachidic  acid  was  extracted  repeatedly  with  ether  to 
remove  the  lower  bromides.  The  ether  mother  liquors  from 
this  extraction  were  concentrated  to  500  cc.  and  cooled  to  0°C. 
A  white  precipitate  settled  out.  This  was  separated  by  filtration 
and  will  be  referred  to  as  Fraction  I. 


290  Fatty  Acids  of  Liver  Lecithin 

The  ethereal  solution  was  evaporated  to  dryness  under  dimin- 
ished pressure  and  cooled  to  0°C.  Two  oily  layers  formed.  These 
were  separated  and  dissolved  separately  in  methyl  alcohol.  Each 
on  cooling  formed  a  sticky  sediment.  These  were  separated. 
The  one  from  the  upper  layer  will  be  referred  to  as  Fraction 
II,  that  from  the  lower  as  Fraction  III. 

The  methyl  alcohol  mother  liquors  from  Fractions  II  and  III 
were  then  combined  and  reduced  with  zinc  dust  and  hydrochloric 
acid  as  described  above.  The  resulting  material  weighed  30  gm. 
It  gave  an  iodine  value  as  follows: 

0.2776  gm.  substance  absorbed  0.296  gm.  iodine  by  the  Wijs  method. 
Ci8H34O2.    Calculated.    Iodine  number   90. 
Found.  "  "      107. 

10  gm.  of  this  material  were  reduced  with  hydrogen  by  Paal's 
method  and  recrystallized  twice  from  dry  acetone.  In  order  to 
decompose  any  methyl  ester  which  might  have  formed  during  the 
zinc  reduction  the  material  was  saponified  with  NaOH  in  methyl 
alcohol  solution.  The  soap  was  precipitated  in  acetone,  dried, 
and  converted  into  free  acid  with  hydrochloric  acid  in  the  presence 
of  ether.  The  ether  solution,  freed  from  inorganic  material  by 
shaking  with  water,  was  dried  and  evaporated.  After  three  more 
recrystallizations  from  dry  acetone  the  acid  analyzed  as  follows: 

Analysis  33.    0. 0994  gm.  substance:  0. 1154  gm.H2O  and  0.2776  gm.  CO2. 
CigH^Oj.    Calculated.    C  75.93,  H  12.76. 
Found.  "  76.15,  "  12.99. 

Melting  point  of  stearic  acid    70-71  °C. 
Found.         68°C. 

Attempt  to  Isolate  Other  Unsaturated  Acids  than  Arachidonic  and 
Oleic  Acids. — Fraction  I,  referred  to  above,  was  a  grayish  brown 
dry  powder,  difficulty  soluble  in  ether.  For  purification  it  was 
extracted  with  hot  methyl  alcohol  and  filtered  hot.  The  insoluble 
material  analyzed  as  follows: 

Analysis  21.    0.1062  gm.  substance:  0. 0374 gm.H2O  and 0. 1030 gm.  CO2. 

0.2041     "  "         :0.3030gm.  AgBr. 

Ci8HsoO2Br4.    Calculated.    C  28.49,  H  3.99,  Br  63.26. 

C20H82O2Br8.  "  25.43,  "  3.42,    "  67.72. 

Found.  "  26.46,  "  4.04,    "  65.26. 


P.  A.  Levene  and  H.  S.  Simms  291 

The  alcohol  extracted  only  a  small  portion  of  the  material. 
When  this  extract  was  cooled  to  20°C.  a  precipitate  formed  which 
was  too  small  for  further  treatment.  On  cooling  the  mother 
liquor  to  —  5°C.  a  second  precipitate  formed.  This  was  separated 
and  analyzed. 

Analysis  22.    0.1062  gm.  substance:  0.0408 gm.H2O  and 0.1 108  gm.  COi 

0.2026    "  "         :0.3018gm.  AgBr. 

Ci8H3oO2Br6.    Calculated.    C  28.49,  H  3.99,  Br  63.26. 
Found.  "  28.45,  "  4.29,    "    63.39. 

In  a  melting  point  determination  this  material  softened  at 
130°C.,  darkening  up  to  170°C.  when  it  partially  melted  and 
partially  decomposed.  At  240°C.  it  became  liquid  and  decom- 
posed in  the  manner  of  octobromarachidic  acid. 

Since  the  analysis  of  the  material  approached  that  of  a  hexa- 
bromide-,  the  substance  was  extracted  six  additional  times  with 
a  large  volume  of  methyl  alcohol.  In  a  melting  point  determina- 
tion the  residue  became  black  at  200°C.  and  decomposed  at 
230°C.  The  combined  extracts  on  cooling  to  0°C.  formed  a  pre- 
cipitate which  analyzed  as  follows: 

Analysis  31.    0.2016  gm.  substance:  0.3146  gm.  AgBr. 
C18H3oO2Br6.    Calculated.    Br63.26. 
C2oH32O2Br8.  "  "   67.72. 

Found.  "   66.41. 

In  a  melting  point  determination  this  material  softened  at 
140°C.,  darkening  with  rise  of  temperature,  and  melting  with 
decomposition  at  243°C. 

The  methyl  alcohol  mother  liquors  were  concentrated  to  100 
cc.  and  cooled  to  0°C.  A  second  precipitate  formed  which  melted 
as  follows.  It  softened  at  140°C.,  partially  decomposed  at  180°C., 
and  melted  with  decomposition  at  243°C. 

Thus  each  fraction  obtained  from  Fraction  I  on  purification  ap- 
proached the  character  of  the  octobromide,  hence  it  is  justifiable 
to  assume  that  this  fraction  consisted  mainly  of  the  octobromide. 

It  was  next  attempted  to  isolate  a  tetrabromide  from  the 
material  referred  to  as  Fractions  II  and  III  (page  290).  Each 
was  dissolved  in  excess  of  ether  and  treated  with  5  parts  of  gasoline. 

Precipitates  were  formed  in  each  and  were  separated.  These 
had  a  sticky  consistency  which  indicated  that  they  were  not  pure. 


292  Fatty  Acids  of  Liver  Lecithin 

They  were  obtained  in  a  quantity  too  small  for  analysis.  The 
combined  gasoline  mother  liquors  were  concentrated  to  small 
volume  and  again  treated  with  gasoline.  A  precipitate  was 
formed  which  analyzed  as  follows: 

Analysis  25.    0.1003  gm.  substance:  0. 0408  gm.  H2O  and  0.1118  gm.CO2. 

0.1972  "  "  :0.2806gm.  AgBr. 

Ci8H32O2Br4.  Calculated.  C  36.01,  H  5.38,  Br  53.28. 
C18H,0O2Br6.  "  "  28.49,  "  3.99,  "  63.26. 

Found.  "  30.39,  "  4.55,    "  60.56. 

The  mother  liquors  from  this  precipitate  were  again  concen- 
trated, dissolved  in  dry  methyl  alcohol,  and  reduced  with  zinc 
dust  and  hydrochloric  acid.  The  reduced  material  had  an  iodine 
number  as  follows: 

0.1990  gm.  substance  absorbed  0.264  gm.  iodine  by  the  Wijs  method. 
Found.     Iodine  number  123. 

The  reduced  material  was  dissolved  in  glacial  acetic  acid  and 
rebrominated.  This  bromine  will  be  referred  to  as  Fraction 
IV. 

The  acetic  acid  solution  was  concentrated  to  dryness  under 
diminished  pressure.  The  residue  was  dissolved  in  hot  absolute 
alcohol  and  cooled  to  —  5°C.  A  precipitate  formed  which  was 
again  dissolved  in  hot  absolute  alcohol  and  reprecipitated  by 
cooling  to  —  5°C.  After  reprecipitating  eight  times  from  absolute 
alcohol  and  once  from  methyl  alcohol,  a  melting  point  determina- 
tion was  made.  It  softened  at  150°C.  and  melted  with  decom- 
position at  240°C.  Apparently  this  is  an  impure  octobromide. 

The  mother  liquors  were  concentrated  and  cooled  to  —  5°C.  A 
precipitate  was  formed  which,  after  several  reprecipitations  from 
absolute  alcohol  and  from  methyl  alcohol,  melted  as  follows: 
At  130°C.  it  softened  and  became  black.  At  230°C.  it  melted  with 
decomposition.  This  also  appears  to  be  an  impure  octobro- 
mide. 

In  order  to  ascertain  whether  a  tetrabromide  was  present  in  the 
mother  liquors  from  the  purification  of  the  last  substance  they 
were  concentrated  to  dryness  under  diminished  pressure  and  dis- 
solved in  gasoline.  Since  no  precipitate  formed  even  on  cooling 
to  —  5°C.,  it  was  concluded  that  no  tetrabromide  was-present. 


P.  A.  Levene  and  H.  S.  Simms  293 

The  three  precipitates  obtained  from  Fractions  II  and  III  were 
combined  and  also  reduced  with  zinc  dust  and  hydrochloric  acid. 
The  reduced  material  gave  an  iodine  number  as  follows: 

0.2815  gm.  substance  absorbed  0.488  gm.  iodine  by  the  Wijs  method. 
Found.    Iodine  number  180. 

This  material  was  also  rebrominated  in  glacial  acetic  acid 
solution.  It  will  be  referred  to  as  Fraction  V.  The  bromide 
solution  was  treated  in  the  same  way  as  Fraction  IV.  It  yielded 
similar  results.  Purification  of  the  insoluble  material  gave  an 
octobromide. 

Fraction  B,  the  Fraction  Soluble  in  Acetic  Acid. 

This  consists  of  the  acetic  acid  mother  liquors  from  which 
Fraction  A  was  filtered.  The  liquors  were  concentrated  under 
diminished  pressure  and  treated  with  gasoline.  An  oily  layer 
settled  out.  This  was  separated,  dissolved  in  methyl  alcohol, 
and  reduced  with  zinc  and  hydrochloric  acid.  20  gm.  of  reduced 
material  were  formed  having  an  iodine  number  as  follows : 

0.1974  gm.  substance  absorbed  0.356  gm.  iodine  by  the  Wijs  method. 
Found.    Iodine  number  180. 

The  acids  were  rebrominated  in  glacial  acetic  acid.  These 
bromides  (Fraction  VI)  were  treated  in  the  same  manner  as 
Fractions  IV  and  V  and  yielded  similar  material.  Had  there 
been  any  tetrabromides  they  would  have  been  found  in  Fractions 
IV,  V,  and  VI. 

Lecithin  Containing  a  Smaller  Percentage  of  Highly  Unsaturated 

Acids. 

A  sample  of  lecithin  was  obtained  from  the  alcohol-insoluble 
portion  of  the  ether  extracts  of  liver.  This  was  purified  by 
dissolving  in  acetic  acid  and  treating  with  alcohol.  The  solu- 
tion after  being  separated  from  the  precipitate  which  formed 
was  concentrated  under  diminished  pressure,  emulsified  with 
water,  and  precipitated  with  acetone.  This  precipitate  was 
dissolved  in  alcohol  and  cooled  to  0°C.  A  precipitate  formed. 
The  solution  was  separated,  concentrated  under  diminished 
pressure,  dissolved  in  ether,  and  precipitated  with  acetone. 


294  Fatty  Acids  of  Liver  Lecithin 

This  precipitate  was  converted  into  the  cadmium  chloride 
salt  and  purified  once  by  the  ether  crystallization  method.  The 
purified  compound  was  converted  into  free  lecithin  by  means  of 
ammonia  in  methyl  alcohol  solution.  The  lecithin  was  again 
converted  into  the  cadmium  chloride  salt  and  again  purified  by 
the  ether  crystallization  process,  using  a  large  volume  of  ether. 
This  material  was  free  from  amino  nitrogen. 

About  1.0  gm.  of  material  was  dissolved  in  15  cc.  of  glacial  acetic  acid. 

2  cc.  of  this  solution:  no  N  (Van  Slyke). 

5  "    "    "  "      :  1.35  cc.  0.1  N  HC1  (Kjeldahl). 

This  cadmium  chloride  salt  of  lecithin  was  then  hydrolyzed  with 
10  per  cent  HC1.  The  iodine  number  was  determined  on  the  fatty 
acids  after  they  had  been  freed  from  mineral  matter  and  dried. 

0.2120  gm.  substance  absorbed  0.256  gm.  iodine  by  the  Wijs  method. 
Found.    Iodine  number  65. 

These  acids  were  then  converted  into  the  lead  salts  and  extracted 
with  ether.  The  ether-soluble  lead  salts  were  converted  into 
free  acids  with  HC1.  Their  iodine  number  was  determined. 

0.2672  gm.  substance  absorbed  0.363  gm.  iodine  by  the  Wijs  method. 
Found.    Iodine  number  136. 


Vita. 

HENRY  S.  SIMMS  was  born  in  South  Manchester,  Connecticut, 
on  May  26,  1896.  He  attended  public  schools  in  Middlebury, 
Vermont,  and  Braintree,  Massachusetts.  He  attended  the 
Braintree  High  School  for  two  years  and  graduated  from  the 
high  school  of  Gorham,  Maine. 

In  1914  he  entered  the  University  of  Maine  where  he  studied' 
chemistry  for  two  years.  After  an  interim  of  one  year,  he  con- 
tinued his  studies  there  for  another  year.  In  1918  he  trans- 
ferred to  the  Massachusetts  Institute  of  Technology  where  he 
studied  for  two  years,  acting  as  assistant  in  the  department  of 
Inorganic  Chemistry  during  the  latter  year. 

In  1920  he  received  the  degree  of  Bachelor  of  Science  from  the 
Massachusetts  Institute  of  Technology.  As  partial  fulfillment  of 
the  requirements  for  this  degree  he  submitted  a  thesis  which  has 
since  appeared  in  two  publications: 

Microanalytical  Methods  in  Oil  Analysis. 

Augustus  H.  Gill  and  Henry  S.  Simms, 

Jour.  Ind.  and  Eng.  Chem.,  1921,  xiii,  547. 
and 
Refractive  Indices  of  Oils, 

Henry  S.  Simms, 

Jour.  Ind.  and  Eng.  Chem.,  1921,  xiii,  546. 

In  1920  he  became  a  Fellow  of  The  Rockefeller  Institute  for 
Medical  Research,  where,  under  the  direction  of  Dr.  P.  A.  Levene, 
he  began  his  research  on  lipoids. 


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FEB  20  1932 
4Pfi  10,935 

JUL  24  1939 
MAR  24  1940 
APR  30  1941 


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THE  WAVERLY  PRE 
BALTIMORE,  U.  ».  A. 


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